This proposal describes an innovative research approach aimed at ultimately revealing the molecular mechanisms that produce and maintain nicotine dependence. To accomplish this, we propose to develop new and generalizable investigative tools by combining cutting-edge features in emerging transgenic and proteomic technologies, specifically by using novel pharmatope-tagged alpha-3-subunit-containing nicotinic receptors in an existing novel knock-in mouse to harness the powerful and sophisticated capabilities of modern mass spectrometry . This project is a new venture in our lab that seeks to apply the fundamental insights gained from our previous structural and functional work to outstanding problems in the drug addiction area. A detailed understanding of nicotine-induced alterations in intracellular protein-protein interactions (i.e., the interactome) involving the putative regulatory cytoplasmic domain of neuronal nicotinic acetylcholine receptors (nAChRs) promises to provide uniquely valuable insights into how behavioral manifestations of nicotine reinforcement, tolerance and sensitization develop. The core concept is that novel pharmatope-tagged knock-in mice can provide general tools to study regulatory proteins interacting with the unusually large cytoplasmic loop that is a feature common to all nAChR subtypes. Our alpha3-knock-in introduces an alpha-bungarotoxin (Bgtx) binding site into neuronal nAChRs that are normally Bgtx- insensitive. The ability to bind Bgtx will be used to biochemically isolate the receptor under mild conditions, together with associated regulatory signaling complexes. These associated proteins will be identified using tandem mass spectrometry and their phosphorylation sites functionally mapped. To critically test this approach, we will focus on the Bgtx-sensitive alphas subunit expressed in primary cultures of superior cervical ganglionic neurons whose post-synaptic alpha3-containing nAChRs are essential for synaptic transmission. This system will allow us to probe the effects of nicotine exposure on the nAChR interactome profile, and will pave the way for studies of alpha3 signaling complexes in the CNS; in particular, in the ventral tegmental area and medial habenula, regions strongly implicated in nicotine addiction. Public Health Relatedness: Knowledge of the cascade of cellular processes and protein interactions regulated by nicotine will contribute to the development of new health interventions. [unreadable] [unreadable]